Hypocrates is a genetically encoded fluorescent biosensor for (pseudo)hypohalous acids and their derivatives

The lack of tools to monitor the dynamics of (pseudo)hypohalous acids in live cells and tissues hinders a better understanding of inflammatory processes. Here we present a fluorescent genetically encoded biosensor, Hypocrates, for the visualization of (pseudo)hypohalous acids and their derivatives. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from Escherichia coli. We show that Hypocrates is ratiometric, reversible, and responds to its analytes in the 106 M−1s−1 range. Solving the Hypocrates X-ray structure provided insights into its sensing mechanism, allowing determination of the spatial organization in this circularly permuted fluorescent protein-based redox probe. We exemplify its applicability by imaging hypohalous stress in bacteria phagocytosed by primary neutrophils. Finally, we demonstrate that Hypocrates can be utilized in combination with HyPerRed for the simultaneous visualization of (pseudo)hypohalous acids and hydrogen peroxide dynamics in a zebrafish tail fin injury model.

A Sensitive Electrochemical Sensor Based on Sonogel-Carbon Material Enriched with Gold Nanoparticles for Melatonin Determination

In this work, the development of an electrochemical sensor for melatonin determination is presented. The sensor was based on Sonogel-Carbon electrode material (SNGCE) and Au nanoparticles (AuNPs). The low-cost and environmentally friendly SNGCE material was prepared by the ultrasound-assisted sonogel method. AuNPs were prepared by a chemical route and narrow size distribution was obtained. The electrochemical characterization of the SNGCE/AuNP sensor was carried out by cyclic voltammetry in the presence of a redox probe. The analytical performance of the SNGCE/AuNP sensor in terms of linear response range, repeatability, selectivity, and limit of detection was investigated. The optimized SNGCE/AuNP sensor displayed a low detection limit of 8.4 nM melatonin in synthetic samples assessed by means of the amperometry technique. The potential use of the proposed sensor in real sample analysis and the anti-matrix capability were assessed by a recovery study of melatonin detection in human peripheral blood serum with good accuracy.

Measuring Practical Reversibility of Surface-Bound DNA for Mechanistic Insight into Folding-Based Sensors

In this paper, we characterize the mass-transport-limited response of surface-tethered redox moieties via flexible DNA linkers using measured voltammetric peak current and peak potential splitting. We demonstrate that peak splitting can be used to differentiate between reversible, quasi-reversible, and irreversible electrochemical regimes of the tethered redox molecule. Interestingly, the transition from one regime to another is dependent on the length and structure of the DNA probe. For example, as the probe length increases the transition from reversible to quasi-reversible occurs at lower scan rates. Additionally, we directly compare the dependence of the peak splitting and peak current as a function of scan rate for ssDNA, dsDNA, and other structured nucleic acids such as stem-loop and pseudoknot probes. Lastly, we find that by interrogating our surfaces with cyclic voltammetry we can observe quantitative differences in the peak splitting once the aptamer is in a bound state and correlate this to the extent of conformational change the sequence undergoes. The observations reported herein are consistent with the postulation that signaling in this class of sensor architectures is dictated by changes in nucleic acid structure and flexibility, which controls the mass transfer rate of the redox probe to the surface of the electrode.

Characterization of graphite-epoxy composite electrodes for free electrochemical detection of adenine and guanine in DNA

Graphite-epoxy composites (GECs) are alternative construction materials for electro­chemical sensors. For these materials, the electron transfer rate constant of some redox reaction depends additionally on the stoichiometric relationship between the insulating and conducting phases of the composite. In this work, the influence of dif­fe­rent ratios of araldite/hardener/graphite on the electrochemical properties of GEC electrodes is evaluated for the simultaneous determination of adenine and guanine in the single chain DNA, using the square wave voltammetry technique. Six GEC electro­des were prepared with different ratios of components, and electrochemically charac­terized by cyclic voltammetry in the presence of ferri/ferrocyanide redox couple as a redox probe. GEC electrodes that showed the best electrochemical responses of redox probe were characterized by thermogravimetric analysis (TGA) and used for the simul­taneous determination of free adenine and guanine in a solution, and DNA oligonu­cle­otides. The best results were obtained for GEC electrodes containing twice higher volu­me of araldite resin with respect to the hardener. TGA analysis revealed presence of 15-26 % of resin for these GEC electrodes. The obtained results revealed potential appl­ication of these GEC electrodes as DNA sensors based on the oxidation signal of guanine.

Sequence-Independent DNA Adsorption on Few-Layered Oxygen-Functionalized Graphene Electrodes: An Electrochemical Study for Biosensing Application

DNA is strongly adsorbed on oxidized graphene surfaces in the presence of divalent cations. Here, we studied the effect of DNA adsorption on electrochemical charge transfer at few-layered, oxygen-functionalized graphene (GOx) electrodes. DNA adsorption on the inkjet-printed GOx electrodes caused amplified current response from ferro/ferricyanide redox probe at concentration range 1 aM–10 nM in differential pulse voltammetry. We studied a number of variables that may affect the current response of the interface: sequence type, conformation, concentration, length, and ionic strength. Later, we showed a proof-of-concept DNA biosensing application, which is free from chemical immobilization of the probe and sensitive at attomolar concentration regime. We propose that GOx electrodes promise a low-cost solution to fabricate a highly sensitive platform for label-free and chemisorption-free DNA biosensing.

A simple label-free electrochemical sensor for sensitive detection of alpha-fetoprotein based on specific aptamer immobilized platinum nanoparticles/carboxylated-graphene oxide

A label-free electrochemical aptamer-based sensor has been fabricated for alpha-fetoprotein (AFP) detection. Platinum nanoparticles on carboxylated-graphene oxide (PtNPs/GO-COOH) modified screen-printed graphene-carbon paste electrode (SPGE) was utilized as an immobilization platform, and the AFP aptamer was employed as a bio-recognition element. The synthesized GO-COOH helps to increase the surface area and amounts of the immobilized aptamer. Subsequently, PtNPs are decorated on GO-COOH to enhance electrical conductivity and an oxidation current of the hydroquinone electrochemical probe. The aptamer selectively interacts with AFP, causing a decrease in the peak current of the hydroquinone because the binding biomolecules on the electrode surface hinder the electron transfer of the redox probe. Effects of aptamer concentration and AFP incubation time were studied, and the current changes of the redox probe before and after AFP binding were investigated by square wave voltammetry. The developed aptasensor provides a linear range from 3.0–30 ng mL−1 with a detection limit of 1.22 ng mL−1. Moreover, the aptamer immobilized electrode offers high selectivity to AFP molecules, good stability, and sensitive determination of AFP in human serum samples with high recoveries.